The long-term research objective of this laboratory is to understand in detail, the molecular mechanisms of Norwalk virus genome expression and replication. Norwalk virus (NV) is the prototype strain of non-cultivable human caliciviruses that are the most important cause of epidemic outbreaks of acute gastroenteritis in humans. NV is considered an emerging virus, as new data based on more sensitive molecular assays indicate that infections with these viruses are more widespread than originally recognized. NV is an exclusively human pathogen, but other caliciviruses infect a broad range of animals and cause persistent, chronic and sometimes lethal infections in their hosts. The ability of the caliciviruses to persist and the potential for emergence, both in prevalence and pathogenesis, exemplify the importance of understanding the replication strategies of the virus at the molecular level. This application proposed studies to understand the viral protein functions critical for replication of the NV genome, and interactions with cellular proteins that regulate these functions. The specific aims of this proposal are 1) To understand the mechanisms of synthesis and processing of the NV non-structural proteins. The details of polyprotein synthesis and post- translation processing will be studied by expression of NV RNA in intestinal cell-free extracts. 2) To understand the mechanisms of control of NV genome expression in intestinal cells. NV non-structural protein synthesis and processing will be investigated in transfected intestinal cells expressing the ORF1 polyprotein. 3) To understand the functional interactions of NV non-structural proteins. Specific interactions of non-structural proteins with RNA will be studied in intestinal cells expressing ORF1. NV and other caliciviruses are unique among animal viruses in structure and genome organization. Thus, detailed dissection of the functions of the NV non-structural proteins likely will lead to elucidation of new mechanisms of RNA virus genome expression.